Transgenic Research最新文献

M-LP/Mpv17L (Mpv17-like protein) is an atypical cyclic nucleotide phosphodiesterase (PDE) without the molecular structure characteristic of the PDE family. Deficiency of M-LP/Mpv17L in mice has been found to result in development of β-cell hyperplasia and improved glucose tolerance. Here, we report another phenotype observed in M-LP/Mpv17L-knockout (KO) mice: afferent cardiac hypertrophy. Although the hearts of M-LP/Mpv17L-KO mice did not differ in size from those of wild-type mice, there was marked narrowing of the left ventricular lumen and thickening of the ventricular wall. The diameter and cross-sectional area of cardiomyocytes in 8-month-old M-LP/Mpv17L-KO mice were increased 1.16-fold and 1.35-fold, respectively, relative to control mice, but showed no obvious abnormalities of cell structure, fibrosis or impaired cardiac function. In 80-day-old KO mice, the expression of hypertrophic marker genes, brain natriuretic peptide (BNF), actin alpha cardiac muscle 1 (ACTC1) and actin alpha 1 skeletal muscle (ACTA1), as well as the Wnt/β-catenin pathway target genes, lymphoid enhancer-binding factor-1 (LEF1), axis inhibition protein 2 (AXIN2) and transcription factor 7 (TCF7), was significantly up-regulated relative to control mice, whereas fibrosis-related genes such as fibronectin 1 (FN1) and connective tissue growth factor (CTGF) were down-regulated. Western blot analysis revealed increased phosphorylation of molecules downstream of the cAMP/PKA signaling pathway, such as β-catenin, ryanodine receptor 2 (RyR2), phospholamban (PLN) and troponin I (cTnI), as well as members of the MEK1-ERK1/2 signaling pathway, which is strongly involved in afferent cardiac hypertrophy. Taken together, these findings indicate that M-LP/Mpv17L is one of the PDEs actively functioning in the heart and that deficiency of M-LP/Mpv17L in mice promotes physiological cardiac hypertrophy.

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO₂ assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD⁺-linked malic enzyme (NAD⁺-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.

The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.

Fundamental to the safety assessment of genetically modified (GM) crops is the concept of negligible risk for newly expressed proteins for which there is a history of safe use. Although this simple concept has been stated in international and regional guidance for assessing the risk of newly expressed proteins in GM crops, its full implementation by regulatory authorities has been lacking. As a result, safety studies are often repeated at a significant expenditure of resources by developers, study results are repeatedly reviewed by regulators, and animals are sacrificed needlessly to complete redundant animal toxicity studies. This situation is illustrated using the example of the selectable marker phosphomannose isomerase (PMI) for which familiarity has been established. Reviewed is the history of safe use for PMI and predictable results of newly conducted safety studies including bioinformatic comparisons, resistance to digestion, and acute toxicity that were repeated to gain regulatory reapproval of PMI expressed from constructs in recently developed GM maize. As expected, the results of these newly repeated hazard-identification and characterization studies for PMI indicate negligible risk. PMI expressed in recently developed GM crops provides an opportunity to use the concept of familiarity by regulatory authorities to reduce risk-disproportionate regulation of these new events and lessen the resulting waste of both developer and regulator resources, as well as eliminate unnecessary animal testing. This would also correctly imply that familiar proteins like PMI have negligible risk. Together, such modernization of regulations would benefit society through enabling broader and faster access to needed technologies.

The presence and levels of transgenic maize in Mexico and the effect this could have on local landraces or closely related species such as teosinte has been the subject of several previous reports, some showing contrasting results. Cultural, social and political factors all affect maize cultivation in Mexico and although since 1998 there has been a moratorium on the commercial cultivation of transgenic maize, Mexico imports maize, mainly from the USA where transgenic cultivars are widely grown. Additionally extensive migration between rural areas in Mexico and the USA and customs of seed exchange between farmers may also play an unintentional role in the establishment of transgenic seed. A comprehensive study of all Mexican maize landraces throughout the country is not feasible, however this report presents data based on analysis of 3204 maize accessions obtained from the central region of Mexico (where permits have never been authorized for cultivation of transgenic maize) and the northern region (where for a short period authorization for experimental plots was granted). The results of the study confirm that transgenes are present in all the geographical areas sampled and were more common in germplasm obtained in the northern region. However, there was no evidence that regions where field trials had been authorized showed higher levels of transgene presence or that the morphology of seed lots harboring transgenic material was significantly modified in favor of expected transgenic phenotypes.

Mice are the most widely used mammalian animal model worldwide. Their use presents many advantages, including our ability to manipulate their genome. Unfortunately, transgenic mice often need to be introgressed to transfer the transgene of interest in a specific mouse line. This time-consuming process can be shortened using the speed congenics technique. However, the need for a panel of informative markers to evaluate the proportion of donor and receiver genomes in different individuals produced at each generation hinders the utilisation of speed congenics. In this study, we present 255 microsatellites and 10 RFLPs which can be used in 18 marker panels, allowing the easy and fast introgression of genes of interest from three mouse lines commonly used for transgenesis (C57BL/6, 129/Sv and FVB) to six mouse lines relevant for biomedical research (BALB/c, C3H, DBA/1, DBA/2, SJL and SWR/J). In addition, our markers analysis confirmed a recently described lack of isogeny in well-established inbred mouse lines available from commercial breeders.

β1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis ^a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of β1,3-galactose and α1,4-fucose by individual β1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis ^a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing β1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis ^a epitope formation.

Insecticidal transgenes, when incorporated and expressed in plants, confer resistance against insects by producing several products having insecticidal properties. Protease inhibitors, lectins, amylase inhibitors, and chitinase genes are associated with the natural defenses developed by plants to counter insect attacks. Several toxin genes are also derived from spiders and scorpions for protection against insects. Bacillus thuringiensis Berliner is a microbial source of insecticidal toxins. Several methods have facilitated the large-scale production of transgenic plants. Bt-derived cry, cyt, vip, and sip genes, plant-derived genes such as lectins, protease inhibitors, and alpha-amylase inhibitors, insect cell wall-degrading enzymes like chitinase and some proteins like arcelins, plant defensins, and ribosome-inactivating proteins have been successfully utilized to impart resistance to insects. Besides, transgenic plants expressing double-stranded RNA have been developed with enhanced resistance. However, the long-term effects of transgenes on insect resistance, the environment, and human health must be thoroughly investigated before they are made available for commercial planting. In this chapter, the present status, prospects, and future scope of transgenes for insect pest management have been summarized and discussed.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.